Erythropoietin (EPO) is a glycoprotein hormone primarly synthesized in the kidney and is the chief regulator of red blood cell production in the body. Commercially available human EPO is produced via recombinant DNA techniques and is known as recombinant human EPO (rhEPO). rhEPO has a molecular mass of approximately 36,000 Daltons, as determined by SDS-PAGE. The molecular mass of the protein backbone is 18,398 Daltons, which indicates that the entire molecule is heavily glycolsylated. The carbohydrate residues are important for in vivo biologic activity.
Maintaining proteins, such as rhEPO, in their native state in aqueous solution or in solid phase is a major challenge to those working in the field of pharmaceutical formulations. The existence of a protein in its native state depends on protein concentration, temperature and nature of solvent, ionic strength of the buffer, etc. Changes in any of these parameters can affect the stability of a protein in solution or solid phase.
Commercial preparations of rhEPO are presently sold as either dilute aqueous solutions or in a lyophilized form which is used to form a dilute aqueous solution, both of which are administered to the body by injection. The concentration of rhEPO in these preparations is very low and the rhEPO is cleared from the body fairly quickly after administration. In view of this limitation of present preparations, there is a need for concentrated preparations of rhEPO, e.g., those containing higher amounts of rhEPO, which can be used in alternate drug delivery systems. We used spray drying techniques to prepare such preparations.
The spray drying of pharmaceuticals is known in the art. For example, see Broadhead, J. et al., "The Spray Drying of Pharmaceuticals," in Drug Dev. Ind. Pharm, 18 (11 & 12), 1169-1206 (1992). In addition to small molecule pharmaceuticals, a variety of biological materials have been spray dried and these include: enzymes, sera, plasma, micro-organisms and yeasts. Spray drying is a useful technique because it can convert a liquid pharmaceutical preparation into a fine, dustless or agglomerated powder in a one-step process. The basic technique comprises the following four steps:
a) atomization of the feed solution into a spray;
b) spray-air contact;
c) drying of the spray; and
d) separation of the dried product from the drying air.
Although known in the field of pharmaceuticals, there has not been much use of spray drying for therapeutic proteins, such as rhEPO. One apparent reason for this is the concern that such proteins may be thermally degraded by the high temperatures utilized in the spray drying process. This is especially true of complex glycoproteins, such as rhEPO, which, in addition to their polypeptide backbones, also have complex branched carbohydrate portions that are required for biological activity. The availability of lyophilization as a ready alternative further steered workers in the field away from using spray drying for therapeutic proteins. However, spray drying provides certain advantages over lyophilization in that it is more economical, fast and easy to scale up. Also, spray dried powders are often more amenable to further processing than lyophilized powders.
It is usually impractical to design formulations based merely on the lyophilization of the bulk drug. This is so because many polypeptides are relatively unstable when lyophilized in low concentrations and they can adsorb to product packaging and lose activity. In order to overcome these problems, many lyophilized pharmaceutical compositions rely on the use of solid diluents, cryoprotectants or bulking agents to increase the amount of solid material present during the lyophilization process. As a result the final lyophilized material contains a small percentage (w/w) of active drug mixed with a large percentage of other solid material.
In contrast, the present invention provides a method for producing an rhEPO powder from bulk rhEPO wherein the powder produced is pure or essentially pure rhEPO or has a higher percentage (w/w) of rhEPO than can be prepared using traditional lyophilization techniques.